An effective universal protocol for the extraction of fructooligosaccharide from the different agricultural byproducts

Alternative bio-refinery technologies are required to promote the commercial utilization of plant biomass components. The fructooligosaccharide (FOS) obtained after hydrolysis of the hemicellulose fractions was mainly applied in the pharmaceutical and food industries. Agricultural bi-product is a rich constituent in dietary fibres, which have prebiotic effects on the intestinal microbiota and the host. Herein we explored the impact of FOS on microbiota modulation and the gut homeostasis effect. High fructooligosaccharide recovery was obtained using alkaline extraction techniques. The enzymatic method produced fructooligosaccharides with minor contamination from fructan and glucan components, although it had a low yield. But combining the alkaline and enzymatic process provides a higher yield ratio and purity of fructooligosaccharides. The structure of the fructooligosaccharide was confirmed, according to FTIR, 13C NMR, 1H NMR and 2D-NMR data. Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods, which were found to be a cost-effective way to boost bio-refining value. Fructooligosaccharides with varying yields, purity, and structure can be obtained.


Description of protocol
The main focus of the new protocol is on the alkaline and enzymatic combined extraction-procedure for water-soluble oligosaccharide (kestose) from banana peel, quantification of hydrolyzed polysaccharide using high-performance liquid chromatography (HPLC-RID), further structural characterization of fructoligosacharide (FOS) using Fourier Transform Infrared (FTIR) spectroscopy, Nuclear Magnetic Resonance ((SEM).

Extraction and manufacture of oligosaccharides from banana peel
There is an increasing interest in using agricultural byproducts to find renewable and economically functional raw material sources in various application fields. Agricultural byproducts have nutrients that can be utilized to grow microorganisms that can create valuable enzymes, complex polysaccharides, and crude proteins. The following are examples of agricultural byproducts high in sucrose and can be utilized to make FOS. A similar argument might be made for peeled fruit such as mango, orange, pineapple, or some baguettes, as well as sugarcane, agave, corn, coconut, cassava, and any discarded leaves [14] . In this study, banana peel was used. After drying in an oven at 300 g of fresh banana peel, 50 g of banana powder was obtained using a grinder. Then, after degreasing by the Soxhlet method, a sample was obtained, treated with -amylase and glucoamylase, centrifuged, and an enzymetreated banana peel sample was obtained. Then, 99% ethanol was added, the precipitate was formed, lyophilized, and finally, about 20 g of a sample was obtained ( Fig. 1 ).

Quantification of kestose (GF2) from BPOE using high-performance liquid chromatography with refractive index detector (HPLC-RID)
In this study, the oligosaccharides were extracted from the peel, a byproduct of bananas, and used as a prebiotic material, kestose (GF2) has good probiotic efficacy among various types of fructooligosaccharides, was used as a marker. It was analyzed using HPLC-RID from the oligosaccharide extract (BPOE) isolated from the banana peel extract. As in Fig. 2 , it was confirmed that BPOE was separated into a total of 5 peaks in the analytical chromatogram. When compared with the RT value (retention time) of the standard GF2, it was confirmed that the peak separated at RT 5.835 min was GF2 contained in BPOE. RT values of the remaining four peaks were 6.482, 9.966, 12.304, and 15.146 min, respectively, and there were unidentified oligosaccharides. Similar studies reported using HPLC, GF2 was identified at the exact location in the yacon extract, and GF3 and GF4 were detected. Therefore, this study focused on GF2, known for its activity as a banana peel oligosaccharide extract prebiotic material, but the remaining GF3 and GF4 were not performed.

Strategy for separation and purification of FOS
Traditional approaches are challenging to identify and separate FOSs due to their structural complexity, high polarity, and difficulty of detection. Furthermore, HSCCC separation is difficult because of their high hydrophilicity, which causes the target compounds to remain in the lower phase, even in n-butanol-water solvent systems. To comply with the HSCCC separation standards, a novel method for FOS preparative separation was used, in which the polarities of FOSs were lowered using acetylation. The compounds were then deacetylated, and FOS separation was confirmed ( Fig. 3 ).   The acetylated kestose (GF2) was chromatographed using the HSCCC method. The following solvent systems were used: petroleum ethern-butanol-methanol-water (3:2:1:4, v/v); stationary phase was in the upper phase; mobile phase was in the lower phase; flow rate was 2.0 mL/min; revolution speed was 800 rpm; retention of the stationary phase was 53.0 percent; sample load was 1 g, and ELSD did detection.

Selection of pre-column derivatization method
The suitable pre-column derivatization method was applied, resulting in ether-forming groups, which can be removed easily after the separation process. According to previous reports [15] , the standard derivatization methods for hydroxyl groups are methyl, acetyl, and phenyl. In our research, the HSCCC was operated after the pre-column derivatization of FOS. Acetylation of hydroxyl groups in FOSs was chosen after weighing the experiment's difficulty, the amount of time it took to complete, and the extent of hydroxyl group substitutions.

High-speed counter-current chromatography (HSCCC) separation and other conditions for separating kestose (GF2)
The selection of an appropriate two-phase solvent solution, which attained a basis for the efficient separation of the target compounds using HSCCC, requires an ideal range of KD values for the employed material, according to the KD value applied in a 0.5 to 2 range based on two-phase solvent. Accordingly, in this study, GF2 was isolated from the banana peel oligosaccharide extract using HSCCC, and a substance deduced as GF2, was collected at an elution time of 5.748. In this work, the FOS (Kestose) structures underwent acetylation. Based on knowledge gained from low-polar compound separation, a variety of solvent systems comprised of petroleum ether, n-butanol, methanol, and water were created with varying volume ratios (3:2:2:3, 3:2:1:4, and v/v) to generate the best KD for the target compounds. The hydroxyls became acetyls after acetylation, and their polarity was significantly lowered. The KD values were evaluated for the GF 2 combination ( Fig. 3 ).

Determination of partition coefficient -distribution constant (KD)
The KD is less than 0.5; the solutes were eluted in close proximity to one another and the solvent front. The KD values of the two-phase solvent systems, including ethyl petroleum ether-n-butanol-methanol-water (3:2:2:3, v/v), were less than 0.5, resulting in The general anomeric-carbon and primary CH2OH carbon assignments. these are tentative; the assignment of primes to numbers is intended mainly to differentiate between resonances of nuclei occupying similar positions specific identification. 'Measured in Deuterium oxide . 'Data compared from Refs. [1 , 2] and from data base (Biological magnetic resonance data bank).
poor peak resolution. The solutes eluted close to and near the solvent front in the two-phase solvent systems utilized for separation. The target compounds were isolated using two-phase solvent systems, but the separation time was too long. The two-phase solvent systems constituted of petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v), with KD values between 0.5 and 2, suitable for separating the target chemicals. As a result, the HSCCC separation was done using the two solvent systems mentioned above. When two-phase solvent systems of petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v).

High-performance liquid chromatography coupled with evaporative light scattering detector HPLC-ELSD analysis to confirm the purified kestose (GF2) form BPOE
HPLC-ELSD analyzed the collected fractions of HSCCC. HPLC was applied to analyze the structure of the target compounds; FOS HPLC optimization. The design of the target compound was analyzed using HPLC, and the ELSD detector was chosen because of its excellent sensitivity and quick application time [16] . Samples eluted with solvent in the C18 chromatographic column have low resolution due to the strong polarity of FOS. The Xamide 100A column was chosen over the Inertsil NH2 Capillary Column for its high resolution and quick processing time. Several elution techniques, including isocratic acetonitrile-water and methanol-water elution, were investigated). The target compounds achieved satisfactory separation when the mobile phase comprised acetonitrile: water (80:20, v/v). The evaporating temperature was 80 °C, the Neb temperature was 90 °C, and the gas flow rate was 1.2 SLM. The column temperature was 30 °C; the mobile phase flow rate was 1.0 mL/min, the evaporating temperature was 80 °C, and the Neb temperature was 90 °C. As a result, a total of 24.9 mg of 1-kestose (GF2) compound was obtained from 1 g of sample, and the final fructooligosaccharide was confirmed to be kestose (GF2) as a result of HPLC-ELSD analysis ( Fig. 3 ).

C-NMR
The 13 C NMR spectrum of HSCCC fraction was shown in Fig. 4 . The spectra of 1-kestose are shown in the figure. These molecules were used as reference materials, all of which contain ᵟ (2 → 1) linkages and are considered the smallest possible fructans and the structural building blocks of larger inulins (oligo-fructans). The 1-kestose carbon signals were assigned compared to reference materials and the previously reported chemical shifts of other 1-kestose [17] .
The fructan spectrum exhibits the same broad signal at 62.28 that was regarded to be strong support for a (2 6) connection of D-Fruf moieties in pure 1-kestose from banana peel oligosaccharide extract [18] . Resonance for C-2 of -D-Fruf of 1-kestose has been set at 103.68 ppm, and an upfield shift of -0.1 ppm has been associated with the elongation of this kind of inulin [18] . The signals are absent in the regions from 105.0, which have been associated with C-2 of a 2,6-di-Osubstituted Fruf unit. The lack of signals at ä 102.3 and 98.9 for C-2 indicates the absence of â-D-Fruf as a reducing moiety.
The 13C NMR spectrum of sucrose in Fig. 4 shows an anomeric region with a signal at 92.45 ppm due to C-1 of an R-D-Glcp moiety. This signal is weaker than the anomeric C-2 of â-D-Fruf ( ᵟ 103.21) in a relative ratio of 1:1. This ratio is observed in all reference materials. It changes pretty drastically once an overlapping of many ketoanomeric signals occurs. The region from ᵟ 60.05 to 62.28 'Data compared from Ref. [1] , and from data base (Biological magnetic resonance data bank).
comprises signals due to C-1 and C-6 of â-D-Fruf residues that sometimes are overlapped. The amount of a-D-Fruf in 1-kestose was found to be 60.85 ppm, which helps explain the C-1 and C6 signals in the pure 1-kestose from banana peel oligosaccharide extract spectrum (60.05). Resonance at 72.54 has also been linked to a Fruf residue at the end.
The absence of an anomeric signal of Fruf linked to internal glucose, the ᵟ 72.54 resonance in the purified extract might be attributed to an internal 2 → 1) Fruf residue. The resonance downfield at ᵟ 81.17 is assigned to a (2 →6) linkage or branched â-D Fruf residues; thus, ᵟ 81.17 in the purified extract spectrum could be due to either -D-Fruf moiety. Another significant region for fructan structural elucidation is located from ᵟ 69.80 to 81.17, where C-5 signals are found. The HSCCC fraction presents two strong signals, one at ᵟ 81.1727 and the other at 81.0821 ppm. According to Lopez et al. [19] the former could be attributed to (2 → 1) linkages and the latter to a (2 → 6) linkage. The C-5 of the -D-Fruf residues linked through O-6 and the residue without a substituent at O-6 could cause these resonances [18] . A prior assignment by Sims et al. [18] demonstrating a structural resemblance of 1-kestose is consistent with the difference of 0.9 ppm between them. This shows that the structure of the pure banana peel extract might be comparable.

1H NMR
The 1 H NMR spectrum of HSCCC fraction is shown in Fig. 5 . The spectra of 1-kestose are shown in the same figure. The unambiguous assignment of H-l was a significant aspect in the entire 1H chemical shift assignment of 1. In the 1 D spectrum, its signal was the sole immediately distinguishable peak. H-2, H-3, H-4, and H-5 are the 'H signals. Because both H-l and H-3 are connected to H-2, the spectrum of 1, cross-peaks between them was seen. The previous designations were supported by the discovery of cross-peaks between the H-3 and H-5 atoms of the two D-fructosyl units and between the H-4 and H-6 atoms of the two D-fructosyl units.
Proton NMR of fructans has been less studied than 13 C NMR, resulting in very few reports on oligofructose being available [20] . Fig. 5 represents the 1 H NMR spectrum of purified bannan peel extract. The chemical shifts for the H-1 of the R-D-Glcp residue can be found in the reference compounds of the kestose type at a value of -5.34. (55; 58; 59). According to these data, the resonance of H-1 fructan seems to agree very well with the chemical shift of internal glucose. The rest of the proton signals have been reported to appear in a narrow region between ᵟ 3.37 and 4.201. Proton assignments are very complex; however, their integration provides relevant information on the length of a polymer. Based on the proton integration of fructan ( Fig. 5 .), it can be established that the purified 1-kestose (fructooligosaccharides) from the banana peel (HSCCC fraction) is constituted of at least 16 residues; therefore, the glucose/fructose ratio is 1-15 at least.

Two-dimension (2D)-homonuclear (H-H) J-resolved -correlation spectroscopy (COSY)
The Overlapping or unresolved peaks made determining the multiplicities of the 'H signals difficult, but the problem was handled by using the homonuclear, J-resolved experiment [21] . The investigation disperses the coupling pattern in the F 1 dimension, allowing exact chemical shifts and signal multiplicity to be determined even for overlapped signals. For example, the H-5 signals of all the sugar residues in 1 were absolutely undetectable in the 1D 'H spectrum but were determined from the J-resolved spectrum ( Fig. 6 ). The coupling patterns made it easier to assign signals that were too close to be distinguished from correlation spectra. Multiple cross-peaks for the H-5 signals have been observed in some correlation tests. Some extra weak peaks in this J-resolved spectrum could indicate the presence of two conformers in the Deuterium Oxide (D 2 O) solution. Multiple signals should, however, be avoided due to the predicted exchange rate.

Particle size analysis (PSA)
The results based on particle size analysis of purified kestose (GF2) are shown in Sup. Fig. 3 . the surface area of the particle sizes value was 6.36 m, and these results showed similar results through TEM analysis. Changes in the morphology and particle size of purified kestose (GF2) may occur due to structural changes due to residues and related substances, which may also affect the functionality of other food uses [22] . In addition, the structural similarity of the purified kestose (GF2) based NMR analysis ( Fig. 6 ), but minor variation was observed in the morphology of purified GF2. In particle size analysis (the state of a sample in the emulsion or powder state of a specific volume) reflects the change in the surface area exposed to moisture, hydration, and viscosity of the polysaccharide are the main factors. There is an inverse relationship between dissolution rate and particle size.

Scanning electron microscopy analysis (SEM)
The purified kestose (GF2) displayed more hygroscopicity and aggregation than standard kestose, according to the microstructure of chicory-derived standard GF2 and purified kestose (GF2). On the other hand, the chicory-derived standard product GF2 has an irregular shape. Still, it is generally spherical, and purified kestose (GF2) has a round shape similar to that of the standard product kestose (GF2) but is highly aggregated on a rough surface. In the case of dried chicory-derived GF2, there were few aggregates, and it was confirmed that it was formed by individual spherical particles, which indicates the adsorption of minor moisture. In powder-dried chicory-derived standard kestose (GF2), the available size is between 29.84-58.54 μm, but there are some larger sizes of 108.56-124.88 μm. On the other hand, the general size of purified kestose (GF2) was 55.34-96.11 μm, which was generally more significant than that of standard kestose (GF2). Toneli et al. [23] evaluated the effect of humidity on the microstructure of spray-dried chicory-derived inulin and observed individual spherical particles. As the humidity increases, the particles begin to agglomerate. The powder becomes a continuous mass when exposed to an ambient environment with an aw (water activity) greater than 0.52. It can no longer be distinguished as individual particles [23] .